Genotyping single nucleotide polymorphisms (SNPs) across species in Old World Monkeys

The development of DNA markers is becoming increasingly useful in the field of primatology for studies on paternity, population history, and biomedical research. In this study, we determine the efficacy of using cross-species amplification to identify single nucleotide polymorphisms (SNPs) in closely related species. The DNA of 93 individuals representing seven Old World Monkey species was analyzed to identify SNPs using cross-species amplification and genotyping. The loci genotyped were 653 SNPs identified and validated in rhesus macaques. Of the 653 loci analyzed, 27% were estimated to be polymorphic in the samples studied. SNPs identified at the same locus among different species (coincident SNPs) were found in six of the seven species studied with longtail macaques exhibiting the highest number of coincident SNPs (84). The distribution of coincident SNPs among species is not biased based on proximity to genes in the samples studied. In addition, the frequency of coincident SNPs is not consistent with expectations based on their phylogenetic relationships. This study demonstrates that cross-species amplification and genotyping using the Illumina Golden Gate Array is a useful method to identify a large number of SNPs in closely related species, although issues with ascertainment bias may limit the type of studies where this method can be applied.     

A High Throughput Single Nucleotide Polymorphism Multiplex Assay for Parentage Assignment in New Zealand Sheep

Accurate pedigree information is critical to animal breeding systems to ensure the highest rate of genetic gain and management of inbreeding. The abundance of available genomic data, together with development of high throughput genotyping platforms, means that single nucleotide polymorphisms (SNPs) are now the DNA marker of choice for genomic selection studies. Furthermore the superior qualities of SNPs compared to microsatellite markers allows for standardization between laboratories; a property that is crucial for developing an international set of markers for traceability studies. The objective of this study was to develop a high throughput SNP assay for use in the New Zealand sheep industry that gives accurate pedigree assignment and will allow a reduction in breeder input over lambing. This required two phases of development- firstly, a method of extracting quality DNA from ear-punch tissue performed in a high throughput cost efficient manner and secondly a SNP assay that has the ability to assign paternity to progeny resulting from mob mating. A likelihood based approach to infer paternity was used where sires with the highest LOD score (log of the ratio of the likelihood given parentage to likelihood given non-parentage) are assigned. An 84 “parentage SNP panel” was developed that assigned, on average, 99% of progeny to a sire in a problem where there were 3,000 progeny from 120 mob mated sires that included numerous half sib sires. In only 6% of those cases was there another sire with at least a 0.02 probability of paternity. Furthermore dam information (either recorded, or by genotyping possible dams) was absent, highlighting the SNP test’s suitability for paternity testing. Utilization of this parentage SNP assay will allow implementation of progeny testing into large commercial farms where the improved accuracy of sire assignment and genetic evaluations will increase genetic gain in the sheep industry.     

Selection and implementation of single nucleotide polymorphism markers for parentage analysis in crossbred cattle population

Crossbreeding is an essential way of improving herd performance. However, frequent parentage record errors appear, which results in the lower accuracy of genetic parameter estimation and genetic evaluation. This study aims to build a single nucleotide polymorphism (SNP) panel with sufficient power for parentage testing in the crossbred population of Simmental and Holstein cattle. The direct sequencing technique in PCR products of pooling DNA along with matrix-assisted laser desorption/ionization time-of-flight MS method for genotyping the individuals was applied. A panel comprising 50 highly informative SNPs for parentage analysis was developed in the crossbred population. The average minor allele frequency for SNPs was 0.43, and the cumulative probability of exclusion for single-parent and both-parent inference met 0.99797 and 0.999999, respectively. The maker-set for parentage verification was then used in a group of 81 trios with aid of the likelihood-based parentage-assignment program of Cervus software. Reconfirmation with on-farm records showed that this 50-SNP system could provide sufficient and reliable information for parentage testing with the parental errors for mother–offspring and sire–offspring being 8.6 and 18.5%, respectively. In conclusion, a set of low-cost and efficient SNPs for the paternity testing in the Simmental and Holstein crossbred population are provided.     

Selection of 29 highly informative InDel markers for human identification and paternity analysis in Chinese Han population by the SNPlex genotyping system

The interest of forensic researchers in single nucleotide polymorphism (SNP) has been attracted because of its potential advantages, such as low mutation rates, amenable to high-throughput automated platform and the improved application in the analysis of degraded samples. In this paper, 29 highly informative insertion/deletion (InDel, a special kind of SNP) markers were selected from the dbSNP () according to the given criteria. 109 unrelated Chinese Han subjects were genotyped for the 29 InDels with SNPlex genotyping system. The allele frequency data revealed that the combined power of discrimination for the 29 InDel markers was 0.999999999990867 and the combined probability of paternity exclusion (PE) was 0.9930. Sensitivity studies were performed to evaluate the flexibility of the SNPlex genotyping system on the set of 29 InDels. Highly reproducible results could be obtained with 40–100 ng genomic DNA and the proportion of total allele drop-in was significantly increased when the amount of DNA added to PCR was lower than 35 ng. These results suggested that the set of 29 InDels was useful in paternity analysis or human identification in the future.     

SNiPORK - a microarray of SNPs in candidate genes potentially associated with pork yield and quality - development and validation in commercial breeds

SNiPORK is an oligonucleotide microarray based on the arrayed primer extension (APEX) technique, allowing genotyping of single nucleotide polymorphisms (SNPs) in genes of interest for pork yield and quality traits. APEX consists of a sequencing reaction primed by an oligonucleotide anchored with its 5' end to a glass slide and terminating one nucleotide before the polymorphic site. Extension with one fluorescently labeled dideoxynucleotide complementary to the template reveals the polymorphism. Ninety SNPs were selected from those associated directly or potentially with pork traits. Of the 90 SNPs, 5 did not produce a positive signal. For 85 SNPs, 100% repeatiblity was proved by double genotyping of 13 randomly chosen boars. In addition, the accuracy of genotyping was verified in 2 sib-families by a Mendelian inheritance of 49-50 homozygous genotypes from sire to sons. Three genotype discrepancies were found (97% accuracy rate). All inaccurities were confirmed by an alternative method (sequencing and PCR-RFLP assays). Moreover, the exclusion power of the chip was evalueted by an SNP inheritance analysis of unrelated boars within each sib-family. In the validation step, 88 boars (13 Pietrain, 31 Landrace, 16 Large White, 8 Duroc, 7 Hampshire x Pietrain crosses, and 13 other hybrid lines) were screened to validate SNPs. Among the 85 selected SNPs, 12 were found to be monoallelic, the rest showing at least two genotypes for the entire population under study. The primary application of the SNiPORK chip is the simultaneous genotyping of dozens of SNPs to study gene interaction and consequently better understand the genetic background of pork yield and quality. The chip may prospectively be used for evolutionary studies, evaluation of genetic distances between wild and domestic pig breeds, traceability tests, as well as the starting point for developing a platform for identification and paternity analysis.     

Impact of genomic selection of AI dairy sires on their likely utilization and methods to estimate fertility: a paradigm shift

Breeding of dairy cattle is undergoing a paradigm shift to genomic selection of potential sires and dams. This undoubtedly will affect how bulls are managed in an artificial insemination (AI) center and impact methods to estimate their ‘fertility'. Our goal is to help decision-makers understand the contents of a straw of semen, current estimates of sire fertility, and how estimates might evolve in a genomic era. Sire fertility is estimated from outcome (pregnant or not) after 300 to > 2,000 inseminations and reported in units (U) as a sire's deviation from a population (> 500 bulls) average pregnancy rate (PR). Too often users do not recognize that imprecision of an estimate encompasses a 3-U range, or more. ‘True fertility' of the sire whose semen is inseminated influences outcome far less than ‘true fertility' of each female and a myriad of microenvironment and management factors. Further, AI centers discard substandard collections and intentionally adjust number of sperm per straw so that differences in pregnancy rates achieved by different sires are minimized! For > 80% of Holstein bulls, estimated ‘sire conception rates' are within a 5.4-U range. In the future, most sires will be 15 to 40 mo old and services will accumulate at > 1,000/mo. Estimated sire conception rates still will be a deviation from the population mean, but should be based on records for the most recent 6 or 12 mo, rather than 48 or 60 mo. Repeated ‘snap shots' every 2 mo would allow AI centers to adjust number of sperm per AI straw from genomic-sires in a timely manner, to maintain high pregnancy rates, and to meet market demands with sires producing ∼40% as many sperm as mature ‘proven sires' of yesteryear.     

Linkage disequilibrium among commonly genotyped SNP variants detected from bull sequence,

Genomic prediction utilizing causal variants could increase selection accuracy above that achieved with SNPs genotyped by currently available arrays used for genomic selection. A number of variants detected from sequencing influential sires are likely to be causal, but noticeable improvements in prediction accuracy using imputed sequence variant genotypes have not been reported. Improvement in accuracy of predicted breeding values may be limited by the accuracy of imputed sequence variants. Using genotypes of SNPs on a high‐density array and non‐synonymous SNPs detected in sequence from influential sires of a multibreed population, results of this examination suggest that linkage disequilibrium between non‐synonymous and array SNPs may be insufficient for accurate imputation from the array to sequence. In contrast to 75% of array SNPs being strongly correlated to another SNP on the array, less than 25% of the non‐synonymous SNPs were strongly correlated to an array SNP. When correlations between non‐synonymous and array SNPs were strong, distances between the SNPs were greater than separation that might be expected based on linkage disequilibrium decay. Consistently near‐perfect whole‐genome linkage disequilibrium between the full array and each non‐synonymous SNP within the sequenced bulls suggests that whole‐genome approaches to infer sequence variants might be more accurate than imputation based on local haplotypes. Opportunity for strong linkage disequilibrium between sequence and array SNPs may be limited by discrepancies in allele frequency distributions, so investigating alternate genotyping approaches and panels providing greater chances of frequency‐matched SNPs strongly correlated to sequence variants is also warranted. Genotypes used for this study are available from https://www.animalgenome.org/repository/pub/ ;USDA2017.0519/.     

Utility and accuracy of template-directed dye-terminator incorporation with fluorescence-polarization detection for genotyping single nucleotide polymorphisms

There are little independent data available about how well single nucleotide polymorphism (SNP) genotyping technologies perform in the typical molecular genetics laboratory. We evaluated the utility and accuracy of a widely used technology, template-directed dye-terminator incorporation with fluorescence-polarization detection (FP-TDI), in a sample of 177 SNPs selected solely on the basis of map location. Genotypes were generated without optimization using standard protocols. Overall, 81% of the SNPs we studied generated readable genotypes by FP-TDI. Thirty-two SNPs were genotyped in duplicate by PCR-RFLP orfluorescent dye-terminator sequencing. Out of a total of 631 duplicate genotypes, no true discrepancies were detected. The true error rate has a 95% chance of lying between 0 and 6 out of 1000 genotypes. We also tested for deviations from Hardy-Weinberg Equilibrium in 33 SNPs genotyped in 50 unrelated individuals, and no significant deviations were detected. Our FP-TDI data were readily adaptable to automated genotype calling using our own method of cluster analysis, which assigns a probability score to each genotype call. We conclude that FP-TDI is both efficient and accurate. The method can easily fill the needs of SNP genotyping projects at the scale typically used for regional or candidate-gene association studies.     

Predictive ability of selected subsets of single nucleotide polymorphisms (SNPs) in a moderately sized dairy cattle population

Several studies have shown that computation of genomic estimated breeding values (GEBV) with accuracies significantly greater than parent average (PA) estimated breeding values (EBVs) requires genotyping of at least several thousand progeny-tested bulls. For all published analyses, GEBV computed from the selected samples of markers have lower or equal accuracy than GEBV derived on the basis of all valid single nucleotide polymorphisms (SNPs). In the current study, we report on four new methods for selection of markers. Milk, fat, protein, somatic cell score, fertility, persistency, herd life and the Israeli selection index were analyzed. The 972 Israeli Holstein bulls genotyped with EBV for milk production traits computed from daughter records in 2012 were assigned into a training set of 844 bulls with progeny test EBV in 2008, and a validation set of 128 young bulls. Numbers of bulls in the two sets varied slightly among the nonproduction traits. In EFF₁₂, SNPs were first selected for each trait based on the effects of each marker on the bulls’ 2012 EBV corrected for effective relationships, as determined by the SNP matrix. EFF₀₈ was the same as EFF₁₂, except that the SNPs were selected on the basis of the 2008 EBV. In DIF_max, the SNPs with the greatest differences in allelic frequency between the bulls in the training and validation sets were selected, whereas in DIF_min the SNPs with the smallest differences were selected. For all methods, the numbers of SNPs retained varied over the range of 300 to 6000. For each trait, except fertility, an optimum number of markers between 800 and 5000 was obtained for EFF₁₂, based on the correlation between the GEBV and current EBV of the validation bulls. For all traits, the difference between the correlation of GEBV and current EBV and the correlation of the PA and current EBV was >0.25. EFF₀₈ was inferior to EFF₁₂, and was generally no better than PA EBV. DIF_max always outperformed DIF_min and generally outperformed EFF₀₈ and PA. Furthermore, GEBV based on DIF_max were generally less biased than PA. It is likely that other methods of SNP selection could improve upon these results.     

MilkProtChip--a microarray of SNPs in candidate genes associated with milk protein biosynthesis--development and validation

MilkProtChip is an oligonucleotide microarray based on the arrayed primer extension (APEX) technique, allowing genotyping of single nucleotide polymorphisms (SNPs) in genes of interest for bovine milk protein biosynthesis. APEX consists of a sequencing reaction primed by an oligonucleotide anchored with its 5'end to a glass slide and terminating one nucleotide before the polymorphic site. The extension with one fluorescently labeled dideoxy nucleotide complementary to the template reveals the polymorphism. A total of 75 SNPs were selected among those associated directly or potentially with milk protein content. Among the 75 SNPs, 4 did not produce a positive signal. Most of the remaining SNPs produced a signal for both strands, except for 4 (one strand). In the validation step, 12 Polish Holstein bulls, 1 Polish Red bull, 1 bison (Bison bonasus), 11 Jersey cows and 25 Polish Holstein cows were screened to validate SNPs. Among the 71 selected SNPs--26 were found monoallelic, the rest showing at least two genotypes for the entire population under study. All the animals were earlier genotyped for 2-5 SNPs by PCR-RFLP and PCR sequencing and all showed complete concordance with APEX genotyping. APEX reactions showed relatively high signal frequencies: more than 0.9, 0.9-0.8 and below 0.8, for 65, 4 and 2 DNA samples, respectively. The primary application of the MilkProtChip is the simultaneous genotyping of dozens of SNPs to reveal and clarify the genetic background of milk protein biosynthesis. The chip may possibly be used for dairy cattle identification and paternity analysis, evolutionary studies, the evaluation of genetic distances between wild and domestic cattle breeds and the domestication history of bovine species.     

Age- and tactic-related paternity success in male African elephants

Information on age- and tactic-related paternity success isessential for understanding the lifetime reproductive strategyof males and constitutes an important component of the fitnesstrade-offs that shape the life-history traits of a species.The degree of reproductive skew impacts the genetic structureof a population and should be considered when developing conservationstrategies for threatened species. The behavior and geneticstructure of species with large reproductive skew may be disproportionatelyimpacted by anthropogenic actions affecting reproductively dominantindividuals. Our results on age- and tactic-specific paternitysuccess in male African elephants are the first from a free-rangingpopulation and demonstrate that paternity success increasesdramatically with age, with the small number of older bullsin the competitive state of musth being the most successfulsires. However, nonmusth males sired 20% of genotyped calves,and 60% of mature bulls (>20 years old) were estimated tohave sired offspring during the 5-year study period. The 3 mostsuccessful males sired less than 20% of the genotyped offspring.Hence, contrary to prediction from behavior and life-historytraits, reproduction was not heavily skewed compared with manyother mammalian systems with a similar breeding system. Nevertheless,these results indicate that trophy hunting and ivory poaching,both of which target older bulls, may have substantial behavioraland genetic effects on elephant populations. In addition, theseresults are critical to the current debate on methods for managingand controlling increasing populations of this species.     

Paternity Tests in Multisired Beef Herds by Blood Grouping

The number of offspring sired by individual bulls in multisired beef herds was determined by blood grouping. Six series of tests, each having from 3 to 18 bulls as possible sires,, yielded from 35 to 114 dam-offspring pairs. The paternity was established for 45 to 90 % of the calves tested in each series. The rest were cases with either more than 1 bull not being excluded as the possible sire, or all bulls excluded from being the sire. The causes for the non-established paternities are discussed. Highly significant differences were found in the number of offspring sired by each bull. In each series, bulls ranking in the top third of the siring order sired 65–100 % of the calves with established paternities. Since a minority of the bulls sired the majority of the calf-crop, the generally recomended number of bulls for herds on the range can perhaps be reduced.     

Selection and use of SNP markers for animal identification and paternity analysis in U.S. beef cattle

DNA marker technology represents a promising means for determining the genetic identity and kinship of an animal. Compared with other types of DNA markers, single nucleotide polymorphisms (SNPs) are attractive because they are abundant, genetically stable, and amenable to high-throughput automated analysis. In cattle, the challenge has been to identify a minimal set of SNPs with sufficient power for use in a variety of popular breeds and crossbred populations. This report describes a set of 32 highly informative SNP markers distributed among 18 autosomes and both sex chromosomes. Informativity of these SNPs in U.S. beef cattle populations was estimated from the distribution of allele and genotype frequencies in two panels: one consisting of 96 purebred sires representing 17 popular breeds, and another with 154 purebred American Angus from six herds in four Midwestern states. Based on frequency data from these panels, the estimated probability that two randomly selected, unrelated individuals will possess identical genotypes for all 32 loci was 2.0 × 10⁻¹³ for multi-breed composite populations and 1.9 × 10⁻¹⁰ for purebred Angus populations. The probability that a randomly chosen candidate sire will be excluded from paternity was estimated to be 99.9% and 99.4% for the same respective populations. The DNA immediately surrounding the 32 target SNPs was sequenced in the 96 sires of the multi-breed panel and found to contain an additional 183 polymorphic sites. Knowledge of these additional sites, together with the 32 target SNPs, allows the design of robust, accurate genotype assays on a variety of high-throughput SNP genotyping platforms.     

Genotyping of Single Nucleotide Polymorphisms in DNA Isolated from Serum Using Sequenom MassARRAY Technology

Background

Large epidemiologic studies have the potential to make valuable contributions to the assessment of gene-environment interactions because they prospectively collected detailed exposure data. Some of these studies, however, have only serum or plasma samples as a low quantity source of DNA.

Methods

We examined whether DNA isolated from serum can be used to reliably and accurately genotype single nucleotide polymorphisms (SNPs) using Sequenom multiplex SNP genotyping technology. We genotyped 81 SNPs using samples from 158 participants in the NYU Women’s Health Study. Each participant had DNA from serum and at least one paired DNA sample isolated from a high quality source of DNA, i.e. clots and/or cell precipitates, for comparison.

Results

We observed that 60 of the 81 SNPs (74%) had high call frequencies (≥95%) using DNA from serum, only slightly lower than the 85% of SNPs with high call frequencies in DNA from clots or cell precipitates. Of the 57 SNPs with high call frequencies for serum, clot, and cell precipitate DNA, 54 (95%) had highly concordant (>98%) genotype calls across all three sample types. High purity was not a critical factor to successful genotyping.

Conclusions

Our results suggest that this multiplex SNP genotyping method can be used reliably on DNA from serum in large-scale epidemiologic studies.     

Impact of candidate sire number and sire relatedness on DNA polymorphism-based measures of exclusion probability and probability of unambiguous parentage

Genetic paternity testing can provide sire identity data for offspring when females have been exposed to multiple males. However, correct paternity assignment can be influenced by factors determined in the laboratory and by size and genetic composition of breeding groups. In the present study, DNA samples from 26 commingled beef bulls and their calves from the Nebraska Reference Herd-1 (NRH1), along with previously reported Illinois Reference/Resource Families data, were used to estimate the impact of sire number and sire relatedness on microsatellite-based paternity testing. Assay performance was measured by exclusion probabilities and probabilities of unambiguous parentage (PUP) were derived. Proportion of calves with unambiguous parentage (PCUP) was also calculated to provide a readily understandable whole-herd measure of unambiguous paternity assignment. For NRH1, theoretical and observed PCUP values were in close agreement (85.3 and 85.8%, respectively) indicating good predictive value. While the qualitative effects on PUP values of altering sire number and sire relatedness were generally predictable, we demonstrate that the impacts of these variables, and their interaction effects, can be large, are non-linear, and are quantitatively distinct for different combinations of sire number and degree of sire relatedness. In view of the potentially complex dynamics and practical consequences of these relationships in both research and animal production settings, we suggest that a priori estimation of the quantitative impact of a given set of interacting breeding group-specific and assay-specific parameters on PUP may be indicated, particularly when candidate sire pools are large, sire relatedness may be high, and/or loci numbers or heterozygosity values may be limiting.     

Prediction of informativeness for microsatellite markers among progeny of sires used for detection of economic trait loci in dairy cattle

Individual loci affecting economic traits can be located using genetic linkage. Application of either daughter or granddaughter designs requires determination of allele origin in the progeny. If only the sires and their progeny are genotyped, the paternal allele origin of progeny having the same genotype as the sire cannot be determined. The expected frequency of informative sons can be predicted for each sire and genetic marker from the allele frequencies in the population. The accuracy of a predictor of the frequency of informative progeny was tested on 103 grandsire x microsatellite combinations. Number of sons per grandsire varied from 24 to 129. Allele frequencies in the population were estimated by genotyping seven sires. The regression of the frequency of informative sons on the predicted frequency was 1.04 with a zero intercept model. Thus, considering the large number of genetic markers available for analysis, predicted informative frequency is a useful criterion for selection of genetic markers.     

Comprehensive paternity assignment: genotype,spatial location and social status in song sparrows,Melospiza Melodia

Comprehensive, accurate paternity assignment is critical to answering numerous questions in evolutionary ecology. Yet, most studies of species with extra‐pair paternity (EPP) fail to assign sires to all offspring. Common limitations include incomplete and biased sampling of offspring and males, particularly with respect to male location and social status, potentially biasing estimated patterns of paternity. Studies that achieve comprehensive sampling and paternity assignment are therefore required. Accordingly, we genotyped virtually all males and >99% of 6‐day‐old offspring over 16 years in a song sparrow (Melospiza melodia) population and used three complementary statistical methodologies to attempt complete paternity assignment for all 2207 offspring. Assignments were highly consistent across maximum likelihood methods that used solely genotype data, and heuristic and integrated Bayesian analyses that included data describing individual locations. Sires were assigned to >99% of all genotyped offspring with ≥95% confidence, revealing an EPP rate of c. 28%. Extra‐pair sires primarily occupied territories neighbouring their extra‐pair offspring; spatial location was therefore highly informative for paternity assignment. EPP was biased towards paired territorial males, although unpaired territorial and floater males sired c. 13% of extra‐pair offspring. Failing to sample and include unpaired males as candidate sires would therefore substantially reduce assignment rates. These analyses demonstrate the integration of genetic and ecological information to achieve comprehensive paternity assignment and direct biological insight, illustrate the potential biases that common forms of incomplete sampling could have on estimated patterns of EPP, and provide an essential basis for understanding the evolutionary causes and consequences of EPP.     

Association between mRNA abundance of functional sperm function proteins and fertility of Holstein bulls

Although the existence of a complex population of mRNA in sperm is well documented, its role has not been completely elucidated. The objective of this study was to determine the relationship of mRNA abundance of sperm specific proteins and sire conception rates (SCR; a fertility index) in Holstein bulls. Samples of sperm from a single collection from commercial Holstein bulls (N = 34) were used to evaluate relative mRNA expression of adenylate kinase (AK) 1, integrin beta (IB) 5, Doppel, nerve growth factor, tissue inhibitors of metalloproteinases (TIMP) 2, lactate dehydrogenase C 1, small nuclear ribonucleoprotein polypeptide N, outer dense fiber 2, and phospholipase C zeta (PLCz) 1 in sperm. With the exception of lactate dehydrogenase C 1 and outer dense fiber 2, the mRNA abundances of these proteins were greater (P < 0.05) for high fertility (> +2 to ≤ 4 SCR) bulls compared with average (≥ 2 to ≤ +2) and low fertility (> −2 to ≤ −4) bulls. Of all the multivariate regression models tested, a combination of AK1, IB5, TIMP2, small nuclear ribonucleoprotein polypeptide N, and PLCz1 accounted for 97.4% of the variance in SCR scores. In the absence of PLCz1, the combination of AK1, IB5, Doppel, nerve growth factor, TIMP, and small nuclear ribonucleoprotein polypeptide N accounted for 96.6% of the variance in SCR scores. In addition, immunocytochemistry confirmed that the sperm-specific protein markers evaluated in this study were present in sperm. In conclusion, frozen-thawed semen from bulls with higher AK1, IB5, TIMP, small nuclear ribonucleoprotein polypeptide N 2 and PLCz1 mRNA abundances in the sperm had greater correlations with sire fertility index and may possess greater probabilities of siring calves.     

An alternative method for genotyping of the <Emphasis Type="Italic">ACE</Emphasis> I/D polymorphism

The mistyping of the angiotensin I-converting enzyme insertion/deletion (ACE I/D) has been well documented, and new methods have been suggested here to improve the genotyping efficiency. Buccal cell samples were collected from 157 young Caucasians, and genotyped using previously known and newly developed PCR amplification genotyping techniques, as well as PCR-RFLP tests for three single nucleotide polymorphisms (rs4327, rs4341 and rs4343). Inconsistent genotyping results were found when using only the PCR amplification genotyping techniques across repeated attempts (8% to 45%), however, individual SNP genotyping was highly consistent (100%). Two SNPs (rs4341 and rs4343) were in complete LD and SNP rs4327 was in high LD with the ACE I/D. The ACE I/D was in HW equilibrium in the portion of the population with consistent genotyping results, whereas the three SNPs were not in HW equilibrium. The mistyping of ACE I/D by only PCR amplification can be improved using alternative methods.